1. Field of the Invention
The present invention relates generally to molecular biology and biochemistry and more specifically to a protein kinase, IxcexaB kinase, which is activated in response to environmental stresses and proinflammatory signals to phosphorylate inhibitors of the NF-xcexaB transcription factors and to methods of using the protein kinase.
2. Background Information
The induction of gene expression due to exposure of a cell to a specific stimulus is a tightly controlled process. Depending on the inducing stimulus, it can be critical to survival of the cell that one or more genes be rapidly induced, such that the expressed gene product can mediate its effect. For example, an inflammatory response stimulated due to an injury to or infection of a tissue results in rapid vasodilation in the area of the injury and infiltration of effector cells such as macrophages. Vasodilation occurs within minutes of the response and is due, in part, to the expression of cytokines in the injured region.
The rapid induction, for example, of an inflammatory response or an immune response, requires that the transcription factors involved in regulating such responses be present in the cell in a form that is amenable to rapid activation. Thus, upon exposure to an inducing stimulus, the response can occur quickly. If, on the other hand, such transcription factors were not already present in a cell in an inactive state, the factors first would have to be synthesized upon exposure to an inducing stimulus, greatly reducing the speed with which a response such as an inflammatory response could occur.
Regulation of the activity of transcription factors involved in such rapid induction of gene expression can occur by various mechanisms. For example, in some cases, a transcription factor that exists in an inactive state in a cell can be activated by a post-translational modification such as phosphorylation on one or more serine, threonine or tyrosine residues. In addition, a transcription factor can be inactive due to an association with a regulatory factor, which, upon exposure to an inducing stimulus, is released from the transcription factor, thereby activating the transcription factor. Alternatively, an inactive transcription factor may have to associate with a second protein in order to have transcriptional activity.
Rarely, as in the case of glucocorticoids, the inducing stimulus interacts directly with the inactive transcription factor, rendering it active and resulting in the induction of gene expression. More often, however, an inducing stimulus initiates the induced response by interacting with a specific receptor present on the cell membrane or by entering the cell and interacting with an intracellular protein. Furthermore, the signal generally is transmitted along a pathway, for example, from the cell membrane to the nucleus, due to a series of interactions of proteins. Such signal transduction pathways allow for the rapid transmission of an extracellular inducing stimulus such that the appropriate gene expression is rapidly induced.
Although the existence of signal transduction pathways has long been recognized and many of the cellular factors involved in such pathways have been described, the pathways responsible for the expression of many critical responses, including the inflammatory response and immune response, have not been completely defined. For example, it is recognized that various inducing stimuli such as bacteria or viruses activate common arms of the immune and inflammatory responses. However, differences in the gene products expressed also are observed, indicating that these stimuli share certain signal transduction pathways but also induce other pathways unique to the inducing stimulus. Furthermore, since inducing agents such as bacteria or viruses initially stimulate different signal transduction pathways, yet induce the expression of common genes, some signal transduction pathways must converge at a point such that the different pathways activate common transcription factors.
A clearer understanding of the proteins involved in such pathways can allow a description, for example, of the mechanism of action of a drug that is known to interfere with the expression of genes regulated by a particular pathway, but the target of which is not known. In addition, the understanding of such pathways can allow the identification of a defect in the pathway that is associated with a disease such as cancer. For example, the altered expression of cell adhesion molecules is associated with the ability of a cancer cell to metastasize. However, the critical proteins involved in the signal transduction pathway leading to expression of cell adhesion molecules have not been identified. Thus, a need exists to identify the proteins involved in signal transduction pathways, particularly those proteins present at the convergence point of different initial pathways that result in the induction, for example, of gene products involved in the inflammatory and immune responses. The present invention satisfies this need and provides related advantages as well.
The present invention provides isolated nucleic acid molecules encoding full length human serine protein kinases, designated IxcexaB kinase (IKK) subunits IKKxcex1 and IKKxcex2. The disclosed IKK subunits share substantial sequence homology and are activated in response to proinflammatory signals to phosphorylate proteins (IxcexaB""s) that inhibit the activity of the NF-xcexaB transcription factor.
For example, the invention provides a nucleic acid molecule having the nucleotide sequence shown as SEQ ID NO: 1, which encodes a cytokine inducible IxcexaB kinase subunit designated IKKxcex1, particularly the sequence shown as nucleotides xe2x88x9235 to 92 in SEQ ID NO: 1, and nucleic acid molecules encoding the amino acid sequence shown as SEQ ID NO: 2, as well as nucleotide sequences complementary thereto. In addition, the invention provides a nucleic acid molecule having the nucleotide sequence shown as SEQ ID NO: 14, which encodes a second cytokine inducible IxcexaB kinase subunit, designated IKKxcex2, and nucleic acid molecules encoding the amino acid sequence shown as SEQ ID NO: 15, as well nucleotide sequences complementary thereto. The invention also provides vectors comprising the nucleic acid molecules of the invention and host cells containing such vectors.
In addition, the invention provides nucleotide sequences that bind to a nucleic acid molecule of the invention, including to nucleotides xe2x88x9235 to 92 as shown in SEQ ID NO: 1. Such nucleotide sequences of the invention are useful as probes, which can be used to identify the presence of a nucleic acid molecule encoding an IKK subunit in a sample, and as antisense molecules, which can be used to inhibit the expression of a nucleic acid molecule encoding an IKK subunit.
The present invention also provides isolated full length human IKK subunits, which can phosphorylate an IxcexaB protein. For example, the invention provides an IKKxcex1 polypeptide having the amino acid sequence shown as SEQ ID NO: 2, particularly the amino acid sequence comprising amino acids 1 to 31 at the N-terminus of the polypeptide of SEQ ID NO: 2. In addition, the invention provides an IKKxcex2 polypeptide having the amino acid sequence shown as SEQ ID NO: 15. The invention also provides peptide portions of an IKK subunit, including, for example, peptide portions comprising one or more contiguous amino acids of the N-terminal amino acids shown as residues 1 to 31 in SEQ ID NO: 2. A peptide portion of an IKK subunit can comprise the kinase domain of the IKK subunit or can comprise a peptide useful for eliciting production of an antibody that specifically binds to an IxcexaB kinase or to the IKK subunit. Accordingly, the invention also provides anti-IKK antibodies that specifically bind to an IKK complex comprising an IKK subunit, particularly to the IKK subunit, for example, to an epitope comprising at least one of the amino acids shown as residues 1 to 31 of SEQ ID NO: 2, and also provides IKK subunit-binding fragments of such antibodies. In addition, the invention provides cell lines producing anti-IKK antibodies or IKK-binding fragments thereof.
The invention also provides isolated IxcexaB kinase complexes. As disclosed herein, an IKK complex can have an apparent molecular mass of about 900 kDa or about 300 kDa. An IKK complex is characterized, in part, in that it comprises an IKKxcex1 subunit, an IKKxcex2 subunit, or both and can phosphorylate an IxcexaB protein.
The present invention further provides methods for isolating an IKK complex or an IKK subunit, as well as methods of identifying an agent that can alter the association of an IKK complex or an IKK subunit with a second protein that associates with the IKK in vitro or in vivo. Such a second protein can be, for example, another IKK subunit; an IxcexaB protein, which is a substrate for IKK activity and is involved in a signal transduction pathway that results in the regulated expression of a gene; a protein that is upstream of the IxcexaB kinase in a signal transduction pathway and regulates IKK activity; or a protein that acts as a regulatory subunit of the IxcexaB kinase or of an IKK subunit and is necessary for full activation of the IKK complex. An agent that alters the association of an IKK subunit with a second protein can be, for example, a peptide, a polypeptide, a peptidomimetic or a small organic molecule. Such agents can be useful for modulating the level of phosphorylation of IxcexaB in a cell, thereby modulating the activity of NF-xcexaB in the cell and the expression of a gene regulated by NF-xcexaB.
The invention also provides methods of identifying proteins that can interact with an IxcexaB kinase, including with an IKK subunit, such proteins which can be a downstream effector of the IKK such as a member of the IxcexaB family of proteins or an upstream activator or a regulatory subunit of an IKK. Such proteins that interact with an IKK complex or the IKK subunit can be isolated, for example, by coprecipitation with the IKK or by using the IKK subunit as a ligand, and can be involved, for example, in tissue specific regulation of NF-xcexaB activation and consequent tissue specific gene expression.